Am J Transl Res 2020;12(9):4902-4922

www.ajtr.org /ISSN:1943-8141/AJTR0110985

Original Article
Chaiqi decoction ameliorates vascular endothelial injury
in metabolic syndrome by upregulating autophagy
Xun Chen1,2*, Xiao-Ru Yan3*, Jing Liu2,4, Li-Ping Zhang2,4
1
Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing, China; 2Beijing University of Chinese
Medicine, 3Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China; 4Dongfang
Hospital of Beijing University of Chinese Medicine, Beijing, China. *Equal contributors and co-first authors.
Received March 17, 2020; Accepted July 15, 2020; Epub September 15, 2020; Published September 30, 2020

Abstract: Objective: The present study aimed to investigate the protective effect of the Chaiqi decoction on vascular
endothelial injury in metabolic syndrome and to determine whether the underlying mechanism was associated with
autophagy. Methods: Chaiqi formula granules were administered to a rat model of metabolic syndrome established
by feeding with a high-salt-sugar-fat diet (HSSFD). The drug-containing serum was used in a hyperglycemia cell
model established using HUVECs cultured with palmitic acid PA. The influence of the Chaiqi decoction on meta-
bolic syndrome-related vascular endothelial injury and autophagy was investigated. Autophagy flux was assessed
in vitro by transfecting cells with GFP-mRFP-LC3 adenoviruses or incubating with DALGreen and DAPRed. Results:
The metabolic syndrome model rats displayed adiposity, hyperglycemia, dyslipidemia, hypertension, thickened in-
tima, deposition of various forms of collagen and lipid droplets, downregulated levels of phosphorylated endothelial
nitric oxide synthase and nitric oxide, upregulated expression of endothelin 1, and dysfunctional autophagy. All
these abnormalities were ameliorated by administration of the Chaiqi decoction to the metabolic syndrome rats.
Furthermore, the Chaiqi-containing serum could upregulate autophagy similarly to rapamycin, in a time-dependent
manner. Conclusion: The Chaiqi decoction could ameliorate vascular endothelial injury by improving autophagy in
metabolic syndrome.

Keywords: Metabolic syndrome, Chaiqi decoction, vascular endothelial injury, autophagy

Introduction ET-1 is a 21 amino acid-long endogenous vaso-

Metabolic syndrome, one of the major risk fac-
tors for cardiovascular disease (CVD), refers to
a group of metabolic disorders that include
symptoms of adiposity, hyperglycemia, dyslipid-
emia, and hypertension [1]. The incidence of
metabolic syndrome has been growing rapidly
over the last decades, and is associated with a
two-fold increase in the incidence of CVD [2, 3].
Unfortunately, CVD is a primary cause of mor-
tality worldwide [4]. Vascular endothelial dys-
function, an early event in the development of
CVD [5], is often accompanied by progression
of metabolic syndrome [6]. In metabolic syn-
drome, the decreased synthesis of nitric oxide
(NO) and notably increased Endothelin-1 (ET-1)
levels indicate vascular endothelial dysfunction
[7, 8]. There is no effective treatment for meta-
bolic syndrome; therefore, improvement of vas-
cular endothelial dysfunction has become a
promising therapeutic strategy to reverse CVD.
constrictor peptide, which is mainly produced
and released in vascular endothelial cells [9].
Under normal physiological conditions, ET-1
binding with endothelin receptor A (ETA) stimu-
lates vasoconstriction, while binding to endo-
thelin receptor B (ETB) causes vasodilation.
Increased ETA and decreased ETB levels lead
to severe vasoconstriction in pathological con-
ditions [10-12]. Many studies have shown
that ET-1 levels in patients with metabolic syn-
drome are higher compared with those in
patients without metabolic syndrome, and is
associated with an increased risk of CVD [13,
14]. Samsamshariat et al., in a cross-sectional
study including 76 participants, reported that
ET-1 was not only increased in patients with
metabolic syndrome, but also suggested that it
is a risk factor for developing endothelial dys-
function [8]. NO, an important vascular signal-
ing molecule, plays a crucial role in regulating
vascular function. NO is synthesized from
 Chaiqi decoction ameliorates vascular endothelial injury
L‑arginine by endothelial-nitric oxide synthase
(eNOS) generating NO and L-citrulline in endo-
thelial cells, and then spreads to the smooth
muscle cells, stimulating them to produce cyclic
(c)GMP and inducing the relaxation of vessels
[15, 16]. Thus, reduced bioavailability and syn-
thesis of NO promotes the endothelial dysfunc-
tion that is commonly associated with CVD and
type 2 diabetes mellitus [17, 18]. Cui et al. dem-
onstrated that endothelial dysfunction with
impaired NO production and eNOS phosphory-
lation was involved in the progression of meta-
bolic syndrome, while endothelial dysfunction
was ameliorated after restoring NO production
and eNOS phosphorylation in metabolic syn-
drome rats [7]. In the present study, we used
ET-1 and NO combined with histological exami-
nation of the thoracic aorta as indicators of
vascular endothelial injury.
Autophagy is a self-protective process that not
only regenerates and releases amino acids,
lipids, and other metabolic precursors to main-
tain cellular homeostasis, but also participates
in the development of a variety of diseases,
such as metabolic syndrome and CVD [19, 20].
Under nutrient excess conditions, autophagy in
CVD tends to be suppressed because of exces-
sive activation of mechanistic target of rapamy-
cin (mTOR) [21, 22], which is a negative regula-
tor of autophagy. In the cardiovascular system,
the accumulation of degraded metabolites
caused by defective autophagy leads to inflam-
mation, apoptosis, and vascular endothelial
dysfunction [22-25]. Thus, there is strong evi-
dence for a causal relationship between
autophagy and the vascular endothelial dys-
function observed in metabolic syndrome.
Traditional Chinese medicine (TCM) plays an
important role in metabolic syndrome treat-
ment and its effects are under investigation.
Chaiqi decoction (Chaiqi), a TCM formula, is
based on the following treatment principles:
“strengthen the spleen and soothe the liver”.
Chaiqi is widely used to treat metabolic syn-
drome in China and has been shown to have
therapeutic effects [26]. Previous studies have
reported that Chaiqi (5.67 g/kg/day to rats, in
distilled water) not only regulates adiposity,
hyperglycemia, dyslipidemia, hypertension, and
other metabolic disorders [27, 28], but also
improves vascular endothelial dysfunction via
the nuclear factor kappa B (NF-κB) signaling
4903 Am J Transl Res 2020;12(9):4902-4922
pathway [29]. Moreover, the NF-κB signaling
pathway can interact with mTOR to regulate
autophagy [30, 31]. Therefore, the present
study aimed to explore the mechanism of
action of Chaiqi in improving metabolic syn-
drome-related vascular endothelial injury
through in vivo and in vitro experiments. The
results showed that Chaiqi could alleviate met-
abolic syndrome-related vascular endothelial
injury via activation of autophagy. This finding
indicated that Chaiqi could be a potential treat-
ment for vascular endothelial injury in metabol-
ic syndrome.
Methods
Chaiqi decoction preparation and materials
Chaiqi formula granules, purchased from China
Resources Sanjiu Pharmaceutical Co. Ltd (Bei-
jing, China), comprised six commonly used
herbs: 10 g Bupleurum (Certificate of quality
number PFC-2018005651), 30 g of Astragalus
membranaceus (Certificate of quality number
PFC-2018005554), 10 g of Citrus aurantiumL
(Certificate of quality number PFC-20180051-
50), 10 g of Atractylodes lancea (Thunb.) DC
rhizome (Certificate of quality number PFC-
2018005335), and 3 g of Panax notoginseng
(Certificate of quality number PFC-20180054-
73). The high-salt-sugar-fat diet (HSSFD, Certi-
ficate of quality number 11002900102121;
50% basal feed, 10% lard, 10% egg yolk pow-
der, 2% cholesterol, 7.5% milk powder, 10%
fructose, 5% Palmitic acid, 3% salt, 2% fish
meal, 0.5% bile salts) was purchased from
Beijing KEAO XIELI FEED Co. Ltd (Beijing, China;
SCXK 2014-0010). Rapamycin (catalog no.
A8167) was purchased from Apexbio (Boston,
MA, USA). Pentobarbital (catalog no. P3636)
and Palmitic acid (PA; catalog no. P0500) were
purchased from Sigma (St. Louis, MO, USA).
D-glucose (catalog no. 8150), bovine serum
albumin (BSA), Free Fatty Acids (catalog no.
A8850), and Dimethyl sulfoxide (catalog no.
D8371) were purchased from Solarbio Life
Science (Beijing, China). Antibodies recognizing
LC3B (Long-chain base protein 3; catalog no.
12741), and beta-actin (catalog no. 4970);
anti-rabbit IgG (catalog no. 4412); and 4’,6-
diamidino-2-phenylindole (DAPI) (catalog no.
8961) were purchased from Cell Signaling
Technology (Danvers, MA, USA). Antibodies
recognizing P62 (sequestome 1 (SQSTM1) cat-
 Chaiqi decoction ameliorates vascular endothelial injury
alog no. ab109012), Beclin 1 (catalog no.
ab207612), ET-1 (catalog no. ab117757), eNOS
(catalog no. ab199956), and Goat Anti-Rabbit
IgG H&L (catalog no. ab97051) were purchas-
ed from Abcam (Cambridge, UK). Antibodies
recognizing phosphorylated (Ser1177) eNOS
(catalog no. 11156-1) were purchased from
SAB Signalway Antibody (Waltham, MA, USA).
Human umbilical vein endothelial cells (HUV-
ECs, catalog no. 8000), endothelial cell medi-
um (ECM; catalog no. 1001), trypsin neutraliza-
tion solution (TNS; catalog no. 0113), 0.25%
EDTA (catalog no. 0103), fetal bovine serum
(FBS, catalog no. 0500), Dulbecco’s Phosphate
Buffered Saline (DPBS, catalog no. 0303) were
purchased from ScienCell Research Labora-
tories (Carlsbad, CA, USA). The enzyme-linked
immunosorbent assay (ELISA) kits for ET-1
(catalog no. DET100) and NO (catalog no.
KGE001) were purchased from R&D Systems
(Minneapolis, MN, USA). Cell Counting Kit 8
(CCK-8, catalog no. HY-K0301), DALGreen (cat-
alog no. D675), DAPRed (catalog no. D677i)
were purchased from Dojindo Molecular Tech-
nologies (Kumamoto, Japan). GFP-mRFP-LC3
(green fluorescent protein-monomeric red fluo-
rescent protein-microtubule associated protein
1 light chain 3 alpha) adenovirus (catalog no.
AP19061002) was purchased from Hanheng
Biotechnology (Shanghai, China).
Animals
The specific pathogen-free (SPF) grade
Sprague-Dawley (SD) rats (n = 53, weighing
200 ± 10 g, 6 to 8-week-old, male) were
obtained from Vital River Laboratory Animal
Technology, Beijing, China (SCXK 2016-0006)
and raised in an SPF animal room at the
Research Institute of Dongfang Hospital (Bei-
jing, China; Certificate number SYXK2019-
0013), which has a temperature of 23-25°C,
humidity of 55 ± 10%, and a light controlled
environment (12-h light/dark cycle), with ad
libitum access to rodent feed and water. The
experimental protocol was approved by the
Animal Welfare Committee of Dongfang
Hospital (Beijing, China; Certificate number
201902). After adaptive feeding for 1 week,
the rats were randomly divided into a control
group (n = 14) and model group (n = 39) for 8
weeks of intervention. The control group was
fed with basic food, while the model group was
fed with the HSSFD. All rats had free access to
4904 Am J Transl Res 2020;12(9):4902-4922
tap water. The rats were weighed once a week.
After 8 weeks of intervention, seven rats were
randomly selected from each group and anes-
thetized using 1% pentobarbital (40 mg/kg) via
intraperitoneal injection. We evaluated the suc-
cess of establishing an animal model of vascu-
lar endothelial injury in metabolic syndrome by
measuring their weight, Lee’s index, body fat
rate, systolic blood pressure (SBP), blood glu-
cose, serum lipid level, serum ET-1 and NO lev-
els; and the results of hematoxylin and eosin
(HE) staining, Masson staining, and Oil Red O
staining of the thoracic aorta. Finally, 28 rats
from the model group (4 rats were eliminated: 2
for underweight and 2 for asphyxiation during
blood pressure measurement) were randomly
divided into four subgroups: The model group (n
= 7), the normal diet group (ND group, n = 7),
the ND+rapamycin (Rapa) group (n = 7), and
the ND+Chaiqi group (n = 7), so that we could
evaluate the effects of diet control and rapamy-
cin or Chaiqi decoction on vascular endothelial
injury in metabolic syndrome. Intervention for 8
weeks comprised: Control group (n = 7), ND
group, ND+Rapa group, ND+Chaiqi group fed
with the basic diet, while the Model group
received HSSFD. Rats in the Control group,
Model group, and ND group were treated by
intragastric administration of distilled water.
Rats in the ND+Rapa group and ND+Chaiqi
group were treated with rapamycin (1 mg/kg/
day, in distilled water) [32] and Chaiqi (5.67 g/
kg/day, in distilled water) [27-29], respectively.
The rats were weighed once a week, and their
body length (naso-anal lengths) and the fat of
epididymis and kidneys were determined at the
end of 8 weeks and 16 weeks. To evaluate the
degree of obesity of the rats, Lee’s index was
calculated as (weight (g)^(1/3))/body length
(cm) [33]. The body fat rate was calculated as
the fat of epididymis and kidneys (g)/weight (g)
× 100%.
Preparation of drug-containing serum of rats
SPF SD male rats (n = 18) were randomly divid-
ed into six groups (3 d control group, 3 d Chaiqi
group, 5 d control group, 5 d Chaiqi group, 7 d
control group, and 7 d Chaiqi group). The rats in
the control group were treated by intragastric
administration of distilled water, while the rats
in the Chaiqi group were treated with Chaiqi
(5.67 g/kg/day, in distilled water) [27-29]. After
3, 5, and 7 d, all groups were anesthetized.
 Chaiqi decoction ameliorates vascular endothelial injury
Blood samples from the six groups were col-
lected separately and sterilely through the
abdominal aorta a 2 h after the last drug admin-
istration. After settling for a while at room tem-
perature, the drug‑containing serum and con-
trol serum were separated by centrifugation at
3000 rpm at 4°C for 15 min and then inacti-
vated at 56°C for 30 min. Finally, the drug-con-
taining serum and control serum were stored at
-80°C after filtration through a 0.22-μm filter to
remove bacteria. Thereafter, the drug-contain-
ing serum was diluted with ECM to of 5%, 10%,
15%, 20%, and 30% when different concentra-
tions were required. The best intervention con-
ditions of the Chaiqi drug-containing serum
were screened using the CCK8 assay.
Cell culture
HUVECs were grown in ECM containing 5%
FBS, 1% ECGs, and 1% Penicillin-Streptomycin.
For hyperglycemic conditions, a proportion of
the HUVECs was grown and passaged in
ECM supplemented with D-glucose to a final
concentration of 25 mM (passage 3-4) accord-
ing to a previously published protocol [25].
Normoglycemic HUVECs were used as the
Control group. When hyperglycemic HUVECs
were 70% confluent, cells were pretreated with
PA (3:1 PA: BSA ratio; PA: 0.4 mM) for 6 h as the
Model group. BSA was used to both stabilize
the insoluble fatty acids and transport them to
the cell. A proportion of the cells was then
exposed to either ECM, rapamycin-ECM (Rapa:
10 μM) or Chaiqi-containing serum-ECM for 24
h (according to the results of CCK8) as the ND
group, ND+Rapa group, ND+Chaiqi group,
respectively. We wanted to evaluate the effects
of nutrient control and rapamycin or Chaiqi
drug-containing serum on vascular endothelial
injury in metabolic syndrome.
Blood biochemical assay
At the end of week 8 and week 16, rats were
fasted overnight and anesthetized using 1%
pentobarbital (40 mg/kg, administered intra-
peritoneally). Blood samples were collected
separately and sterilely through the abdominal
aorta and centrifuged at 3000 rpm for 15 min
to obtain serum for subsequent assays. The
levels of blood glucose, total cholesterol, tri-
glyceride, high density lipoprotein, and low den-
sity lipoprotein were measured using an auto-
matic biochemical analyzer (JAPAN. HITACHI,
Tokyo, Japan, 7180).
4905 Am J Transl Res 2020;12(9):4902-4922
Systolic blood pressure (SBP)
The SBP of rats in all groups was measured at
8 and 16 weeks of the intervention period,
respectively. SBP was determined using the
non-invasive tail-cuff plethysmography method
and recorded using an automatic sphygmoto-
nography instrument (BP-98A, Softron, Beijing,
China).
Detection of ET-1 and NO levels
The cell culture supernatants were collected by
centrifugation at 3000 rpm, at 4°C for 5 min,
and stored at -80°C. Blood samples were col-
lected as mentioned above. The ET-1 and NO
levels were determined using commercially
available ELISA kits according to the protocols
provided by manufacturers.
Histological examination
About 0.5 cm of thoracic aorta tissue was fixed
in 4% paraformaldehyde sectioned, and the
tissue sections were subjected to HE staining,
Masson staining, and Oil Red O staining. We
examined the slides under a light microscope
(200 ×, 400 ×; Olympus IX71, Olympus, Tokyo,
Japan). Three sections were selected and mea-
sured from each group. We then calculated
intima thickness, collagen volume fraction (col-
lagen area/total area × 100%), and lipid vol-
ume fraction (Lipid area/total area × 100%).
Transmission electron microscopy (TEM)
About 1 mm3 of thoracic aorta tissue or 1 × 107
cells were fixed in glutaral+osmium tetrachlo-
ride. The samples were then dehydrated with
acetone solution, followed by embedding and
semi-thin sectioning. Complex dyes (0.25%
sodium borate: 0.5% basic fuchsin 1:1) were
used for staining. After images were acquired
under a microscope, ultrathin sectioning was
carried out and the sections were placed on
thin membrane copper grids prepared with
0.45% Fonnvar solution. Uranyl acete staining
and lead staining were then carried out at room
temperature. The grids were dried using filter
paper and then observed using TEM (TECNAI
G2 SPIRIT, FEI, Holland).
Assess autophagic flux
To assess autophagy flux after different inter-
ventions, a GFP-mRFP-LC3 adenovirus was
 Chaiqi decoction ameliorates vascular endothelial injury
transfected into cultured HUVECs for 5 h at a
multiplicity of infection (MOI) of 100. The GFP-
mRFP-LC3 protein shows both red (mRFP) and
green (GFP) fluorescence at neutral pH. It can
form yellow (red+green) puncta that represent
autophagosome formation. When an autopha-
gosome fuses with a lysosome, the GFP is
degraded because it is pH-labile, whereas
mRFP-LC3 (Red puncta in merge pictures) is
maintained in the puncta, which then repre-
sents the autolysosomes [34]. The relative
ratio of red-only versus yellow puncta is us
ed as an index of autophagy flux. Signals
were visualized under a confocal laser-scan-
ning microscope (Olympus FV1000). Images
were acquired using FV10-ASW3.0 software
(Olympus).
To assess autophagy flux of Chaiqi drug-con-
taining serum dynamically, after intervention,
cells were incubated with DALGreen and
DAPRed in the culture medium for 1 h at 37°C.
Green (DALGreen) represents the autolyso-
somes and Red (DAPRed) represents autopha-
gosomes and autolysosomes [35]. The fluores-
cence images were collected under a fluores-
cence microscope (Olympus IX71).
Immunofluorescence (IF)
Fresh thoracic aorta tissue was fixed in cold
4% formaldehyde and immersed overnight in
buffer containing 30% sucrose. Tissues were
then paraffin-embedded and stored until use.
Sections were cut at 4 µm, adhered to charged
slides, dewaxed with xylene, and hydrated
using an ethanol gradient. After rinsing, sec-
tions were washed three times in phosphate-
buffered saline (PBS) for 5 min, followed by
antigen retrieval. After excess fluid was
removed, the sections were incubated over-
night at 4°C with anti-LC3B antibodies (1:100,
Cell Signal Technology). After several washes
with PBS, the sections were incubated with
Cy3-conjugated rabbit anti-IgG for 60 min at
room temperature and then counterstained
with DAPI. Signals were visualized on a confo-
cal laser-scanning microscope (Olympus
FV1000). Images were acquired using the
FV10-ASW3.0 software. Image analysis was
performed using computerized densitometry
in Image J (Media Cybernetics Inc., Bethesda,
MD, USA).
4906 Am J Transl Res 2020;12(9):4902-4922
CCK-8 assay of cell activity
Both normoglycemic and hyperglycemic cells
were incubated in 96-well plates for 24 h. The
cell density was 1 × 104 cells/well before incu-
bation. The Chaiqi drug-containing serum and
the blank serum was diluted with ECM to a
concentration of 5%, 10%, 15%, 20%, and 30%.
In the experiment, normoglycemic cells were
exposed to ECM as the control group. ECM
without cells was used as a blank group.
Hyperglycemic cells pretreated with PA (0.4 μm)
for 6 h were exposed to different concentra-
tions of Chaiqi-containing or blank serum for
24, 48, and 72 h as experimental groups. Each
group had three duplicate wells. The CCK-8
reagent was added to every well according to
the manufacturer’s instructions and incubated
for 2 h. The cell activity rate was calculated
as the optical density ((OD) value of the experi-
mental well-the OD value of a blank well)/(OD
value of contrast well-OD value of blank well) ×
100%. The OD value was detected using a full-
wavelength micrometer (Syngene H1; Syngene
International, Bangalore, India). The best inter-
vention conditions of the Chaiqi drug-contain-
ing serum should satisfy two requirements: 1,
The OD value should be significant differently
from the OD value of the blank serum at the
same concentration and intervention time; 2,
The cell activity rate should be the closest to
100%, but no more than 100%.
Western blotting analysis
Western blotting was performed as described
previously [36]. Cell lysates and aortic homog-
enates were prepared in Radioimmunopreci-
pitation assay (RIPA) buffer (150 mM NaCl, 1%
Triton X-100, 1% sodium deoxycholate, 50 mM
Tris-HCl pH 7.5, 2 mM EDTA, 1 mM Pmetabolic
syndromeF, 1 mM DTT, 1 mM Na3VO4, and 5
mM NaF) containing a protease inhibitor cock-
tail (Applygen Technologies Inc., Beijing, China)
and a phosphatase inhibitor cocktail (Applygen
Technologies Inc.). Proteins were separated by
SDS-PAGE and transferred to polyvinylidene
fluoride (PVDF) membranes. After blocking
with 5% skim milk or 5% BSA, the membranes
were incubated overnight at 4°C with primary
antibodies recognizing beta‑actin (1:1000),
LC3B (1:1000), P62 (1:5000), phospho-eNOS
(1:1000), eNOS (1:1000), ET-1 (1:5000), and
Beclin 1 (1:2000). After incubation with the
 Chaiqi decoction ameliorates vascular endothelial injury

Reagents (Syngene G:BOX

Chemi XX9, UK). Gray-scale
levels of the immunoreactive
protein bands were quantified
using Image J (Media Cyber-
netics Inc.).

Statistical analysis

All the experiments repeat-

ed at least three times. The
results are expressed as the
mean ± SEM. The Komogorov-
Smirnov test (sample size ≥
5) and q-q map (sample size <
5) were used to analyze
whether the normal distribu-
tion was satisfied. The rank-
sum test was used for inter-
group comparisons that did
not meet the normal distribu-
tion, and Levene’s test was
used to determine the homo-
geneity of variance. The inde-
pendent sample t test was
used for inter-group compari-
sons when the variance was
homogeneous, and the t’ test
Figure 1. Effects of receiving the HSSFD for 8 weeks on SD rats. The com- was used when the variance
parison of (A) weight, (B) Lee’s index, (C) body fat rate, (D) SBP determined
using the non-invasive tail-cuff plethysmography method and recorded with
was not homogeneous. SPSS
an automatic sphygmotonography, (E) Glu and lipid levels measured using 21.0 statistical software (IBM
an automatic biochemical analyzer between the control and model group. Corp., Armonk, NY, USA) was
Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01 and ***P < used for the statistical analy-
0.001 versus the control; n = 7 per group. sis. P < 0.05 was considered
as statistically significant.

Results

HSSFD successfully induced

metabolic syndrome related
vascular endothelium injury
in rats

As shown in Figure 1, the rats

receiving HSSFD for 8 weeks
Figure 2. Effects of receiving the HSSFD for 8 weeks on SD rats. The com- showed markedly increased
parison of serum (A) ET-1 and (B) NO expression measured using ELISA kits weight, Lee’s index, body fat
between the control and model groups. Data are expressed as the mean ± rate, SBP, glucose (Glu), cho-
SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the Control; n = 7
per group.
lesterol (CHO), triglycerides
(TG), low density lipoprotein
cholesterol (LDLC), and decre-
HRP-conjugated secondary antibody (1:20000), ased high density lipoprotein cholesterol (HD-
the immunoreactive proteins were visualized LC) compared with the control rats. Moreover,
using an enhanced chemiluminescence kit as expected, serum ET-1 levels in the model
(ECL kit, Applygen Technologies Inc., Beijing, rats were markedly increased compared with
China) and an Immobilon Western Detection those in the control rats, while serum NO levels

4907 Am J Transl Res 2020;12(9):4902-4922

 Chaiqi decoction ameliorates vascular endothelial injury

lagen and lipid droplets depo-

sition (All P < 0.05. Figure 3B,
3C). Our previous researches
showed that serum VCAM-
1, ICAM-1, IL-6, and TNF-a lev-
els in the model rats were
markedly increased compared
with those in the control rats
[27-29]. Taken together, the
results demonstrated that
administration of the HSSFD
for 8 weeks can induce a rat
model of vascular endothelial
injury in metabolic syndrome.

Chaiqi decoction meliorates

metabolic syndrome related
vascular endothelial injury in
vivo

After another 8 weeks of inter-

vention, compared with the
those of the model group, the
weight, Lee’s index, body fat
rate, SBP, Glu, CHO, TG, and
LDLC were decreased and
HDLC was increased in the
ND, ND+Rapa, and ND+Chaiqi
groups. In terms of weight,
Lee’s index and SBP, the ND+
Rapa and ND+Chaiqi groups
performed better than the ND
group. For TG, the ND+Chaiqi
group performed better than
Figure 3. Histological analysis of the 400 × magnification thoracic aorta in both the ND and ND+Rapa
the control and model groups. (A) In the model group we observed disorga- groups (all P < 0.05. Table 1;
nized endothelial cells, thickened intima, and a variety of collagen and lipid Figure 4). Moreover, as the
droplets deposition by HE staining, Masson staining, and Oil Red O staining,
compared with those of control group. We also observed greater (B) intimal
images of HE staining, Masson
thickness, (C) the deposition area of collagen and lipid droplet in the model staining and Oil Red O stain-
group analyzed by Image J. The blue area presented by Masson staining is ing (Figure 5A) showed, com-
collagen deposition, and the red drops presented by Oil Red O staining are pared with the model group,
lipid droplet depositions. Bar: 20 μm, Data are expressed as the mean ± the thickening of the intima
SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the control; n = 3
per group.
and the area of collagen and
lipid deposition in thoracic
aorta tissue were significant
were greatly reduced (both P < 0.05. Figure 2). ameliorated in the ND+Rapa and ND+Chaiqi
Images of HE staining, Masson staining, and groups, and to a greater extent in the ND+Chaiqi
Oil Red O staining revealed that the control group than in the ND+Rapa group (All P < 0.05).
thoracic aorta tissue had neatly arranged endo- However, there were no significant differences
thelial cells without thickened intima, or mas- between the model group and ND group for
sive collagen and lipid droplet deposition intima thickness (P > 0.05. Figure 5B, 5C).
(Figure 3A). In contrast, the model rats thoracic Furthermore, the ET-1 level in serum was
aorta tissue displayed disorganized endothelial decreased in the ND, ND+Rapa, and ND+Chaiqi
cells, thickened intima, and varied levels of col- groups compared with that in the model group,

4908 Am J Transl Res 2020;12(9):4902-4922

 Chaiqi decoction ameliorates vascular endothelial injury

Table 1. Effects of diet control, rapamycin, and Chaiqi decoction for 8 weeks on weight in rat models
Weight (g)
group
0w 1w 2w 3w 4w 5w 6w 7w 8w
Control 462.71 ± 27.73 464.86 ± 27.99 475.00 ± 27.06 483.43 ± 24.49 487.00 ± 29.95 500.86 ± 29.17 504.86 ± 29.53 505.43 ± 30.60 510.71 ± 24.03
Model 509.29 ± 37.46* 514.14 ± 38.74* 521.14 ± 36.36* 524.86 ± 23.12** 534.43 ± 27.04** 548.71 ± 27.96** 565.43 ± 22.47** 568.57 ± 22.92** 585.14 ± 22.89***
ND 509.57 ± 36.65 *
500.43 ± 35.18 490.29 ± 16.71 &
508.86 ± 19.91 502.71 ± 20.42 &
513.86 ± 24.89 &
517.57 ± 18.65 &&
519.43 ± 21.33 &&
519.43 ± 18.08&&&
ND+Rapa 512.71 ± 37.89 *
501.14 ± 33.14 *
492.29 ± 23.73 493.00 ± 22.35 &
478.14 ± 14.69 &&&
490.86 ± 16.2 &&&
500.00 ± 11.73 &&&
495.14 ± 10.11 &&&
499.71 ± 12.26&&&
ND+Chaiqi 511.71 ± 36.00* 509.00 ± 35.08* 477.29 ± 23.58& 482.29 ± 24.37&& 472.29 ± 23.78&& 486.00 ± 24.59&& 487.86 ± 23.74&&& 478.00 ± 24.36&&& 481.86 ± 26.65&&&
n = 7, *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control; &P < 0.05, &&P < 0.01, &&&P < 0.001 vs. Model.

4909 Am J Transl Res 2020;12(9):4902-4922

 Chaiqi decoction ameliorates vascular endothelial injury

samples (P < 0.05. Figure 6A).

For NO, compared with that in
the model group, the NO level
in serum was increased in the
ND, ND+Rapa, and ND+Chaiqi
groups, with higher levels in
the ND+Chaiqi group com-
pared with those in the ND
and ND+Rapa groups (All P <
0.05. Figure 6D). The trend of
phosphorylation of eNOS in
thoracic aorta tissues were
similar to those in serum,
while there were no significant
differences between the ND+
Chaiqi and ND+Rapa groups
(P > 0.05. Figure 6C). Taken
together, these results show-
ed that the Chaiqi decoction
meliorated metabolic syn-
drome-related vascular endo-
thelial injury in vivo and the
effect was better than that
achieved by rapamycin and
diet control only.

Chaiqi decoction ameliorates

metabolic syndrome related
vascular endothelial injury via
autophagy activation

Rapamycin can suppress the

mTOR signaling pathway and
is an inducer of autophagy
[37]. Therefore, we examined
levels of autophagy-related
factors (LC3, Beclin-1, p62),
and the levels of autophago-
somes and autolysosomes in
Figure 4. Effects of diet control, rapamycin, and Chaiqi decoction for 8 weeks rat thoracic aorta. As shown
on metabolic syndrome rats. (A) weight, (B) Lee’s index, (C) body fat rate, (D) by the TEM images (Figure
SBP was determined using the non-invasive tail-cuff plethysmography meth-
od and recorded using automatic sphygmotonography, (E) Glu and lipid level 7A), autophagosomes and
measured using an automatic biochemical analyzer. Data are expressed as autolysosomes could be rec-
mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 versus the control; ognized in the ND+Rapa and
&P < 0.05, &&P < 0.01, and &&&P < 0.001 versus the Model; #P < 0.05, ND+Chaiqi groups. Further-
##P < 0.01, and ###P < 0.001 versus ND; @P < 0.05, @@P < 0.01, and more, the IF images (Figure
@@@P < 0.001 versus ND+Rapa; n = 7 per group.
7B) showed that compared
with the control group, the
while the ET-1 level was lower in the ND+Rapa integrated optical density of LC3 was decreas-
and ND+Chaiqi groups than in the ND group (All ed in the model and ND groups, while it was
P < 0.05. Figure 6B). The trend of ET-1 levels in increased in the ND+Rapa and ND+Chaiqi
thoracic aorta tissues were similar to that in groups (All P < 0.05. Figure 7C). Data from
serum, while the ET-1 level in the ND+Chaiqi western blotting further confirmed the above-
samples was lower than that in the ND+Rapa mentioned changes in autophagy levels.

4910 Am J Transl Res 2020;12(9):4902-4922

 Chaiqi decoction ameliorates vascular endothelial injury

Figure 5. Histological analysis of the 200 ×/400 × magnification thoracic aorta in the experimental groups. (A)
Representative images of HE staining, Masson staining, and Oil Red O staining of the rat thoracic aorta. Effects of
receiving diet control, rapamycin, and Chaiqi decoction on intimal thickness (B) and the deposition of collagen and
lipid droplets (C), analyzed using Image J. The blue area presented by Masson staining is collagen deposition, and
the red drops presented by Oil Red O staining are lipid droplet deposition. Bar: 20 μm, Data are expressed as the
mean ± SEM. Bar: 50 μm or 20 μm; *P < 0.05, **P < 0.01, and ***P < 0.001 versus the control; &P < 0.05, &&P
< 0.01, and &&&P < 0.001 versus the model; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus the ND; @P < 0.05,
@@P < 0.01, and @@@P < 0.001 versus ND+Rapa; n = 3 per group.

Compared with the control group, the LC3II/I
ratio (a useful marker for autophagy), and
Beclin-1 levels were significantly decreased in
the model and ND groups, while they were
increased in the ND+Rapa and ND+Chaiqi
groups (All P < 0.05. Figure 8A, 8B). p62 (an
autophagy adaptor protein) showed the reverse
pattern (P < 0.05. Figure 8C). There was no
significant difference between the ND+Rapa
group and ND+Chaiqi group for LC3II/I, Beclin-1,
and p62 (All P > 0.05).
Chaiqi drug-containing serum ameliorates
metabolic syndrome-related vascular endothe-
lial injury in vitro
Among the different intervention conditions
whose OD value reached statistical significance

4911 Am J Transl Res 2020;12(9):4902-4922

 Chaiqi decoction ameliorates vascular endothelial injury

Figure 6. The expression of ET-1 and NO in thoracic aorta tissue and serum among the different experimental
groups. Representative images and quantitative analysis of (A) ET-1 expression and (C) phosphorylation of eNOS in
thoracic aorta tissue assessed using western blotting and Image J, respectively (n = 3 per group). The expression of
(B) ET-1 and (D) NO in serum assessed using ELISA (n = 7 per group). Data are expressed as the mean ± SEM. *P
< 0.05, **P < 0.01, and ***P < 0.001 versus the control; &P < 0.05, &&P < 0.01, and &&&P < 0.001 versus the
model; #P <0.05, ##P < 0.01, and ###P < 0.001 versus ND; @P < 0.05, @@P < 0.01, and @@@P < 0.001 versus
ND+Rapa.

compared with each blank group (Table 2),
conditions comprising the Chaiqi drug-contain-
ing serum administered intragastrically for 7
days, intervention for 24 h, and a concentration
of 10% could ameliorate the PA and hypergly-
cemia-induced reduction in cell viability by
99.41%. These condition was used for subse-
quent experiments, because its effect was the
closest to 100%, without exceeding 100%
(Figure 9A). Moreover, we evaluated the effect
of Chaiqi drug-containing serum on ameliorat-
ing vascular endothelial injury by measuring
ET-1 and NO levels. The ET-1 level in cells
increased in the model and ND groups, and
was not significantly different to that in
ND+Rapa group, whereas the ET‑1 level
decreased in the ND+Chaiqi group compared
with that in the control group (Figure 9B). The
trend of ET-1 in cell culture supernatants was
similar to that in cells, in which ND+Chaiqi
group showed a higher ET-1 than that in the
control group; however, the difference did not
reach statistical significance (Figure 9C). The
level of phosphorylated eNOS in cells decreas-
ed in the model group, but was not signifi-
cantly different in the ND, ND+Rapa, and the
ND+Chaiqi groups compared with that in the
control group (Figure 9D), while the p-eNOS
levels in the ND+Rapa and ND+chiqi groups
were higher than that in the ND group. The NO
levels in cell culture supernatants showed
similar trends to the levels of p-eNOS in cells.

4912 Am J Transl Res 2020;12(9):4902-4922

 Chaiqi decoction ameliorates vascular endothelial injury

Chaiqi drug-containing se-

rum ameliorates vascular
endothelial injury in vitro via
autophagy activation

As shown by the TEM images

(Figure 10A), autophagosom-
es and autolysosomes could
be observed in the ND+Rapa
and ND+Chaiqi groups. In
addition, western blotting re-
vealed that, the LC3II/I ratio
were significantly decreased
in the model and ND groups,
but increased in the ND+Rapa
and ND+Chaiqi groups (All P <
0.05. Figure 11A). The trend
of Beclin-1 was similar to the
LC3II/I ratio, while its level in
the ND group was higher than
that in the model group (All P <
0.05. Figure 11B). However,
p62 showed the reverse pat-
tern (All P < 0.05. Figure 11C).
Furthermore, we transfected
a GFP-mRFP-LC3-expressing
adenovirus into cultured HUV-
ECs because it is difficult to
assess autophagy flux in vivo,
especially in vascular tissue.
Images (Figure 10B) and relat-
ed data (Figure 10C) in the
transfected HUVECs further
confirmed the abovemention-
Figure 7. The number of autophagosomes and autolysosomes, and the ex- ed changes in autophagy lev-
pression of LC3 among different experimental groups. (A) Representative
images of transmission electron microscopy of the rat thoracic aorta. The els. Compared with the contr-
single arrows represent autophagosome and the double arrows represent ol group, both autolysosomes
autolysosomes. Bar = 500 nm. Representative image (B) and quantitative (red dots) and autophago-
analysis (C) of LC3 expression in thoracic aorta tissue using immunofluo- somes (yellow dots) were sig-
rescence and Image J analysis, respectively. The blue spots represent cell
nuclei and red puncta represent LC3. LC3 puncta were detected using anti- nificantly decreased in the
LC3 antibodies under a confocal laser scanning microscope. Bar = 10 μm; model group, and increased in
Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < the ND+Rapa and ND+Chaiqi
0.001 versus the control; &P < 0.05, &&P < 0.01, and &&&P < 0.001 versus groups. There were no signifi-
the model; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus ND; @P < 0.05,
@@P < 0.01 and @@@P < 0.001 versus ND+Rapa; n = 3 per group.
cant differences between the
control and ND groups. In
addition, there was no statisti-
There was no significant difference between cal significance between the ND+Rapa and
the ND and model groups, and the ND+Chaiqi ND+Chaiqi groups.
group had higher p-eNOS levels than the
ND+Rapa group (Figure 9E). Taken together, Chaiqi drug-containing serum upregulated au-
these results supported the view that Chaiqi tophagy in a time-dependent manner
drug‑containing serum ameliorates metabolic
syndrome-related vascular endothelial injury in To assess autophagy flux of Chaiqi drug-con-
vitro, to a greater extent than rapamycin and taining serum dynamically, cells after interven-
nutrient control only. tion were preincubated with DALGreen and

4913 Am J Transl Res 2020;12(9):4902-4922

 Chaiqi decoction ameliorates vascular endothelial injury

Figure 8. The expression of autophagy-related proteins in thoracic aorta tissue among the different experimental
groups. Representative image and quantitative analysis of the expression of (A) LC3B, (B) Beclin-1, and (C) p62 by
western blotting and Image J, respectively. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, and
***P < 0.001 versus the control; &P < 0.05, &&P < 0.01, and &&&P < 0.001 versus the model; #P < 0.05, ##P <
0.01, and ###P < 0.001 versus ND; @P < 0.05, @@P < 0.01, and @@@P < 0.001 versus ND+Rapa. n = 3 per group.

Table 2. OD values of different conditions of Chaiqi drug-containing serum intervention in HUVECs

injured by PA and hyperglycemia
OD
group
5% 10% 15% 20% 30%
24 h B 0.75 ± 0.01 0.89 ± 0.02 0.90 ± 0.03 0.87 ± 0.08 0.84 ± 0.04
d3 0.76 ± 0.01 0.89 ± 0.02 0.86 ± 0.02* 0.83 ± 0.03 0.82 ± 0.02
d5 0.90 ± 0.06*** 1.28 ± 0.11*** 0.99 ± 0.15 0.89 ± 0.09 0.90 ± 0.14
d7 0.89 ± 0.05 ***
1.02 ± 0.11 **
0.89 ± 0.09 0.82 ± 0.06 0.92 ± 0.11
48 h B 0.89 ± 0.07 1.22 ± 0.03 1.22 ± 0.02 1.34 ± 0.04 1.22 ± 0.04
d3 0.85 ± 0.03 1.29 ± 0.03*** 1.47 ± 0.11*** 1.38 ± 0.05 1.38 ± 0.12**
d5 1.08 ± 0.05*** 1.54 ± 0.20*** 1.64 ± 0.14*** 1.47 ± 0.20 1.47 ± 0.18**
d7 1.24 ± 0.16 ***
1.54 ± 0.20 ***
1.49 ± 0.16 ***
1.51 ± 0.18 *
1.31 ± 0.13
72 h B 0.96 ± 0.02 1.26 ± 0.14 1.43 ± 0.11 1.64 ± 0.04 1.54 ± 0.04
d3 0.87 ± 0.01*** 1.26 ± 0.14 1.41 ± 0.14 1.76 ± 0.16* 1.56 ± 0.03
d5 1.25 ± 0.13 ***
1.74 ± 0.12 ***
1.80 ± 0.07 ***
1.61 ± 0.12 1.69 ± 0.03***
d7 1.65 ± 0.12 ***
1.71 ± 0.08 ***
1.73 ± 0.12 ***
1.68 ± 0.07***
1.60 ± 0.11
B: Blank serum group; d3, d5, and d7: Chaiqi drug-containing serum administered intragastrically for 3 days, 5
days, and 7 days, respectively. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the blank serum group.

DAPRed to stain autolysosomes and autopha-
gosomes. As shown in Figure 12A, compared
with 0 h, both the green and red fluorescent
areas increased significantly at 12 h, 18 h, and
24 h, and the stained areas increased with
time (Figure 12B). The LC3II/I ratio also
increase with time (Figure 12C). This indicated
that Chaiqi drug-containing serum upregulated
autophagy in a time-dependent manner.
Discussion
The vascular endothelium is an important cel-
lular component of the cardiovascular system
and plays a crucial role in maintaining cardio-
vascular homeostasis. Therefore, endothelial
dysfunction is the one of the earliest detect-
able vascular abnormalities and is predictive of
later cardiovascular complications [38-40].
Animal experiments have provided ample evi-
dence that metabolic syndrome is strongly
associated with vascular endothelial injury.
Feeding with an HSSFD induced abnormalities
of metabolic syndrome-related indices, such as
abdominal fat deposition, abnormal glucose
tolerance, dyslipidemia, hyperinsulinemia, and
hypertension. It also led to vascular endothelial

4914 Am J Transl Res 2020;12(9):4902-4922

 Chaiqi decoction ameliorates vascular endothelial injury

saturated fatty acids) were

nearly three times higher
than in the healthy controls
[47]. Palmitate also inhibited
cell proliferation and induced
apoptosis in mice aortic endo-
thelial cells, which were sepa-
rated from mice fed high-
calorie and high-cholesterol
diets for 3 months [48]. The
lipotoxic effect of PA has been
implicated in the pathogene-
sis of numerous CVDs [49].
In our study, to mimic the
vascular endothelial dysfunc-
tion caused by HSSFD and
the constant exposure of vas-
cular endothelial cells to the
condition of chronic hypergly-
cemia and PA, we utilized a rat
model fed with HSSFD con-
taining 5% PA and a cell model
maintained and passaged in
media containing 25 mM glu-
cose that were further acutely
challenged with 0.4 mM PA
according to a previously pub-
lished protocol [25, 27-29], to
gain a better understanding of
the pathological effects of
vascular endothelial injury in
metabolic syndrome. More-
over, previous studies lack an
evaluation of diet or nutrient
controls. To explore the effect
Figure 9. Screening of Chaiqi drug-containing serum and the levels of ET-1 of diet control in improving
and NO among the different experimental groups. (A) CCK-8 screening show- vascular endothelial injury
ing that he best intervention conditions comprised intragastric administra- in metabolic syndrome, this
tion for 7 days, intervention for 24 h, and a concentration of 10%. Repre-
sentative images and quantitative analysis of (B) ET-1 levels and (D) the
study conducted intervention
phosphorylation of eNOS in cells using western blotting and Image J, respec- on the basis of diet and nutri-
tively. The levels of (C) ET-1 and (E) NO in cell culture supernatants, as as- ent control.
sessed using ELISA. Data are expressed as the mean ± SEM. *P < 0.05, **P
< 0.01, and ***P < 0.001 versus the control; &P < 0.05, &&P < 0.01, and The mammalian target of
&&&P < 0.001 versus the model; #P < 0.05, ##P < 0.01, and ###P < 0.001
versus ND; @P < 0.05, @@P < 0.01, and @@@P < 0.001 versus ND+Rapa. rapamycin (mTOR) is an evolu-
tionarily conserved serine/
threonine kinase that senses
dysfunction via an inflammatory reaction and fluctuations in extracellular and intracellular
oxidative stress, accompanied by increased nutrients to modulate cellular growth, metabo-
ET-1 and decreased of NO levels [41-46]. lism, and survival [50, 51]. Additionally, the
Besides, a clinical study indicated that free mTOR inhibitor rapamycin (Rapa, also known as
fatty acid levels in patients with diabetes were sirolimus) has vital uses in CVDs [52, 53],
50% higher compared with those in healthy oncology [54], and transplantation medicine
controls during overnight fasting, and postpran- [55], especially in metabolic disorders such as
dial levels of palmitate (one of the predominant diabetic nephropathy [56, 57], diabetic retinop-

4915 Am J Transl Res 2020;12(9):4902-4922

 Chaiqi decoction ameliorates vascular endothelial injury

ET-1 levels and the aggrega-

tion of platelets and neutro-
phils, reduce the release of
cellular chemokines and the
migration of vascular smooth
muscle cells to the intimal
layer, and improve intimal
hyperplasia [52]. Moreover,
rapamycin exhibits protective
effects on endothelial cells in
metabolic syndrome by induc-
ing autophagy, reducing apo-
ptosis and endoplasmic retic-
ulum stress [63], improving
the activity of NO and phos-
phorylation of eNOS, and de-
creasing ET-1 levels [53, 64].
In addition, rapamycin signifi-
cantly inhibited glucose and
palmitate treatment-induced
apoptosis and inflammation,
which indicated that the in-
duction of autophagy by rapa-
mycin might have a beneficial
effect on diabetic atheroscle-
rosis [25]. In the present
study, we found that diet and
nutrient control and interven-
tion with rapamycin or the
Chaiqi decoction improved
the abnormalities in metabo-
lic syndrome-related indices
(weight, lee’s index, body fat
Figure 10. The number of autophagosomes and autolysosomes among the rate, SBP, Glu, CHO, TG, HDLC,
different experimental groups. (A) Representative images of transmission and LDLC) and vascular endo-
electron microscopy in cells. The single arrows represent autophagosomes
and the double arrows represent autolysosomes. Bar = 1 μm or 500 nm. To thelial injury-related indices
assess autophagy flux of different interventions, a GFP-mRFP-LC3 adeno- (intimal thickness, collagen,
virus was transfected into cultured HUVECs for 5 h at an MOI of 100. (B) and lipid droplet deposition,
Representative images and (C) quantitative analysis of autophagosome and ET-1, NO, and phosphorylation
autolysosome numbers by manual counting. The yellow puncta represent
autophagosomes and red puncta represent autolysosomes, which were de- of eNOS) in vivo and in vitro.
tected under confocal laser scanning microscope. Bar = 10 μm; Data are Notably, Chaiqi was more eff-
expressed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 ective in reducing the deposi-
versus the control; &P < 0.05, &&P < 0.01, and &&&P < 0.001 versus the tion of collagen and lipid drop-
model; #P < 0.05, ##P < 0.01, and ###P < 0.001 versus ND; @P < 0.05,
@@P < 0.01, and @@@P < 0.001 versus ND+Rapa; n = 3 per group.
lets, increasing NO in serum
and cell culture supernatants,
and decreasing ET-1 in cells,
athy [58], diabetic heart disease [59], and tissue, and cell culture supernatants than
metabolic syndrome [60] by targeting mTOR. rapamycin. Diet and nutrient control was the
Studies have shown that mTOR activity in mice least effective and showed no significant differ-
with type 2 diabetes is significantly increased ence with the model group in terms of intimal
[61], and rapamycin can reduce body weight thickness, cellular ET-1 levels, and NO levels in
and fat mass, and improve insulin resistance serum and cell culture supernatants. These
and blood glucose levels by inhibiting mTOR results indicated that Chaiqi could not only
activity [32, 62]. Besides, it can also decrease improve the abnormalities in metabolic syn-

4916 Am J Transl Res 2020;12(9):4902-4922

 Chaiqi decoction ameliorates vascular endothelial injury

Figure 11. The levels of autophagy-related proteins in cells among the different experimental groups. Representa-
tive images and quantitative analysis of the levels of (A) LC3B, (B) Beclin-1, and (C) p62, assessed using western
blotting and Image J, respectively. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P <
0.001 versus the control; &P < 0.05, &&P < 0.01, and &&&P < 0.001 versus the model; #P < 0.05, ##P < 0.01, and
###P < 0.001 versus ND; @P < 0.05, @@P < 0.01, and @@@P < 0.001 versus ND+Rapa.

Figure 12. Effects of Chaiqi drug-containing serum on autophagy flux in HUVECs injured by PA and hyperglycemia.
To assess autophagy flux of Chaiqi drug-containing serum dynamically, cells after intervention were incubated with
DALGreen and DAPRed in the culture medium for 1 h at 37°C. (A) Representative images were detected by immuno-
fluorescence assay and (B) quantitative analysis of autophagosome and autolysosome expression was performed
using Image J. Green (DALGreen) represents the autolysosomes and red (DAPRed) represents both autolysosomes
and autophagosomes. Bar = 10 μm. Representative images and quantitative analysis of the expression of LC3B (C)
in cells were assessed using western blotting. Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01 and
***P < 0.001 versus 0 h.

4917 Am J Transl Res 2020;12(9):4902-4922

 Chaiqi decoction ameliorates vascular endothelial injury
drome-related indices, but could also amelio-
rate vascular endothelial injury in metabolic
syndrome.
Autophagy is a catabolic process in eukaryotic
cells that relies on lysosomes to degrade old
proteins and dysfunctional organelles, and
participates in cell development, differentia-
tion, and maintenance of homeostasis [65]. In
recent years, autophagy has been shown to
participate in the pathogenesis of various
CVDs, such as cardiomyopathy [66], heart fail-
ure [67], atherosclerosis [68], and myocardial
ischemia/reperfusion injury [69]. In metabolic
syndrome, high glucose and fat status can
increase the production of reactive oxygen spe-
cies and the expression of cell surface adhe-
sion molecules, induce inflammatory changes,
leading to vascular endothelial injury, accompa-
nied by autophagy inhibition, which act as a
protective mechanism against apoptosis [7, 70,
71]. Cui et al. showed that metabolic syndrome
in rats induced by a high fat diet and 10%
fructose water feeding resulted in hyperten-
sion, obesity, metabolic abnormity, insulin
resistance, downregulated p-eNOS levels, vas-
cular endothelial injury, and autophagy dys-
function. All these abnormalities in metabolic
syndrome rats were ameliorated after restoring
autophagy [7]. In a separate study, treatment
of endothelial cells with high concentrations of
glucose and PA impaired basal autophagy,
which resulted in increased cell apoptosis and
inflammation [25]. Collectively, these results
suggested that the induction of an autophagic
response in endothelial cells exerts cytopro-
tective actions under excess nutrient-induced
stress. However, some studies have shown that
glucose and palmitate induce autophagy in
endothelial cells [72-74]. These opposite
effects might depend on concentration, inter-
vention time, and different cell conditions. To
date, various naturally occurring agents, such
as Curcumin [74, 75], Resveratrol [76, 77],
Berberine [78], Royal Jelly [79], and Naringenin
[80], have been reported to have cardiovascu-
lar protective effects via upregulating autopha-
gy in endothelial cells. Interestingly, these men-
tioned above agents are mostly ingredients of
TCM. The effects of Chinese herbal compound
prescription, including Chaiqi decoction, have
not been examined. In the present study, we
found that autophagy was impaired after meta-
bolic syndrome-related vascular endothelial
4918 Am J Transl Res 2020;12(9):4902-4922
injury and diet/nutrient control might not
effectively improve the level of autophagy in
vivo and in vitro. However, Chaiqi could upregu-
late autophagy similarly to the autophagy
inducer rapamycin. Notably, autophagy flux
analysis indicated that there was no statisti-
cally significant difference between Chaiqi and
rapamycin for the count of autophagosomes
and autolysosomes and the levels of autopha-
gy-related proteins. Furthermore, we assessed
autophagy flux of Chaiqi drug-containing serum
dynamically by incubating with DALGreen and
DAPRed and found that Chaiqi drug-containing
serum upregulated autophagy in a time-depen-
dent manner. These results indicated that
Chaiqi could ameliorate vascular endothelial
injury in metabolic syndrome by upregulating
autophagy.
There have been many studies on the regula-
tion of hunger-induced autophagy, involving the
regulation of activity of several kinases, includ-
ing mammalian target of rapamycin Complex 1
(mTORC1), unc-51 like autophagy activating
kinase 1 (ULK1), and Adenosine 5’-monophos-
phate (AMP) activated protein kinase (AMPK)
[81, 82]. Under low nutrient conditions, these
kinases (and other kinases) coordinate the for-
mation of autophagosomes, which are digested
by proteases in lysosomes and break down the
products for subsequent synthesis of new pro-
teins or to fuel cells [83]. However, the autoph-
agy regulation mechanism of endothelial cells
has not been fully determined under high-nutri-
ent states (such as metabolic syndrome) [25].
Under excess nutrient conditions, AMPK activi-
ty and the phosphorylation of AMPK and ULK1
were diminished, accompanied by impaired
basal autophagy [25, 84] in human aortic
endothelial cells. Intriguingly, reactivation of
AMPK with activators AICAR, A769662, or
phenformin failed to restore autophagy, while
the mTOR inhibitor rapamycin significantly
induced autophagy [25]. mTOR is usually acti-
vated under high nutrient conditions, which
would explain that the uncoupling of AMPK
from ULK1 activation under high glucose and
palmitate conditions occurs because AMPK
can no longer access and interact with ULK1
because of mTOR activation. However, in-
creased levels of phosphorylated (Thr389)
p70S6K and the ratio of phosphorylated
(Ser2448) mTOR to total mTOR protein were not
observed [25]. It is possible that the failure to
 Chaiqi decoction ameliorates vascular endothelial injury
detect increased mTOR activity might have
been caused by the lack of a time-course study
under these conditions, because high nutrient
levels lead to mTOR activation [85]. In this
regard, we found that autophagy was sup-
pressed during vascular endothelial injury in
metabolic syndrome. Chaiqi could restore met-
abolic syndrome-induced abnormalities simi-
larly to the mTOR inhibitor rapamycin. Our fur-
ther experiments will investigate whether
Chaiqi decoction could upregulate autophagy
via the mTOR signalling pathway.
Conclusions
In conclusions, our findings demonstrated that
the Chaiqi decoction ameliorated vascular
endothelial injury by enhancing autophagy. Our
study provides additional insights into the clini-
cal application of TCM in metabolic syndrome
and related cardiovascular complications.
Acknowledgements
The study was supported by the self-selected
topic of the Beijing University of Chinese
Medicine (Grant no. 2019-JYB-JS-114).
Disclosure of conflict of interest
None.
Address correspondence to: Dr. Li-Ping Zhang,
Beijing University of Chinese Medicine, No.11
Bei San Huan Dong Lu, Chaoyang District, Beijing
100029, China. E-mail: lpzhang2005@126.com; Dr.
Jing Liu, Dongfang Hospital of Beijing University of
Chinese Medicine, Fangxingyuan, Fangzhuang,
Fengtai District, Beijing 100078, China. E-mail: liu-
jing20120114@163.com
References
[1] Dommermuth R and Ewing K. Metabolic syn-
drome systems thinking in heart disease. Prim
Care 2018; 45: 109-129.
[2] Mottillo S, Filion KB, Genest J, Joseph L, Pilote
L, Poirier P, Rinfret S, Schiffrin EL and Eisenberg
MJ. The metabolic syndrome and cardiovascu-
lar risk a systematic review and meta-analysis.
J Am Coll Cardiol 2010; 56: 1113-1132.
[3] Berwick ZC, Dick GM and Tune JD. Heart of the
matter: coronary dysfunction in metabolic syn-
drome. J Mol Cell Cardiol 2012; 52: 848-56.
[4] Rawshani A, Sattar N, Franzén S, Rawshani A,
Hattersley AT, Svensson AM, Eliasson B and
Gudbjörnsdottir S. Excess mortality and cardio-
vascular disease in young adults with type 1
4919 Am J Transl Res 2020;12(9):4902-4922
diabetes in relation to age at onset: a nation-
wide, register-based cohort study. Lancet
2018; 392: 477-486.
[5] Sitia S, Tomasoni L, Atzeni F, Ambrosio G,
Cordiano C, Catapano A, Tramontana S,
Perticone F, Naccarato P, Camici P, Picano E,
Cortigiani L, Bevilacqua M, Milazzo L, Cusi D,
Barlassina C, Sarzi-Puttini P and Turiel M.
From endothelial dysfunction to atherosclero-
sis. Autoimmun Rev 2010; 9: 830-834.
[6] Tziomalos K, Athyros VG, Karagiannis A and
Mikhailidis DP. Endothelial dysfunction in
metabolic syndrome: prevalence, pathogene-
sis and management. Nutr Metab Cardiovasc
Dis 2010; 20: 140-146.
[7] Cui F, Guan Y, Guo J, Tian YM, Hu HF, Zhang XJ
and Zhang Y. Chronic intermittent hypobaric
hypoxia protects vascular endothelium by ame-
liorating autophagy in metabolic syndrome
rats. Life Sci 2018; 205: 145-154.
[8] Samsamshariat SZA, Sakhaei F, Salehizadeh
L, Keshvari M and Asgary S. Relationship
between resistin, endothelin-1, and flow-medi-
ated dilation in patient with and without
metabolic syndrome. Adv Biomed Res 2019; 8:
16.
[9] Beghetti M, Black SM and Fineman JR. endo-
thelin-1 in congenital heart disease. Pediatr
Res 2005; 57: 16r-20r.
[10] da Silva AA, Kuo JJ, Tallam LS and Hall JE. Role
of endothelin-1 in blood pressure regulation in
a rat model of visceral obesity and hyperten-
sion. Hypertension 2004; 43: 383-387.
[11] Iglarz M, Steiner P, Wanner D, Rey M, Hess P
and Clozel M. Vascular effects of endothelin
receptor antagonists depends on their selec-
tivity for ETA versus ETB receptors and on the
functionality of endothelial ETB receptors. J
Cardiovasc Pharmacol 2015; 66: 332-337.
[12] Mazzuca MQ and Khalil RA. Vascular endothe-
lin receptor type B: structure, function and
dysregulation in vascular disease. Biochem
Pharmacol 2012; 84: 147-162.
[13] Rocha NG, Templeton DL, Greiner JJ, Stauffer
BL and DeSouza CA. Metabolic syndrome and
endothelin-1 mediated vasoconstrictor tone in
overweight/obese adults. Metabolism 2014;
63: 951-956.
[14] Yu AP, Tam BT, Yau WY, Chan KS, Yu SS, Chung
TL and Siu PM. Association of endothelin-1
and matrix metallopeptidase-9 with metabolic
syndrome in middle-aged and older adults.
Diabetol Metab Syndr 2015; 7: 111.
[15] Förstermann U and Münzel T. Endothelial nitric
oxide synthase in vascular disease: from mar-
vel to menace. Circulation 2006; 113: 1708-
1714.
[16] Wyatt AW, Steinert JR and Mann GE.
Modulation of the L-arginine/nitric oxide sig-
 Chaiqi decoction ameliorates vascular endothelial injury
nalling pathway in vascular endothelial cells.
Biochem Soc Symp 2004; 143-156.
[17] Sessa WC. eNOS at a glance. J Cell Sci 2004;
117: 2427-2429.
[18] Gao L, Siu KL, Chalupsky K, Nguyen A, Chen P,
Weintraub NL, Galis Z and Cai H. Role of un-
coupled endothelial nitric oxide synthase in
abdominal aortic aneurysm formation: treat-
ment with folic acid. Hypertension 2012; 59:
158-166.
[19] Choi AM, Ryter SW and Levine B. Autophagy in
human health and disease. N Engl J Med
2013; 368: 651-662.
[20] Tan Y, Gong Y, Dong M, Pei Z and Ren J. Role of
autophagy in inherited metabolic and endo-
crine myopathies. Biochim Biophys Acta Mol
Basis Dis 2019; 1865: 48-55.
[21] Ren J, Sowers JR and Zhang Y. Metabolic
stress, autophagy, and cardiovascular aging:
from pathophysiology to therapeutics. Trends
Endocrinol Metab 2018; 29: 699-711.
[22] Zhang Y, Whaley-Connell AT, Sowers JR and
Ren J. Autophagy as an emerging target in car-
diorenal metabolic disease: from pathophysi-
ology to management. Pharmacol Ther 2018;
191: 1-22.
[23] Ren SY and Xu X. Role of autophagy in meta-
bolic syndrome-associated heart disease.
Biochim Biophys Acta 2015; 1852: 225-231.
[24] Kosacka J, Nowicki M, Paeschke S, Baum P,
Blüher M and Klöting N. Up-regulated autopha-
gy: as a protective factor in adipose tissue of
WOKW rats with metabolic syndrome. Diabetol
Metab Syndr 2018; 10: 13.
[25] Weikel KA, Cacicedo JM, Ruderman NB and
Ido Y. Glucose and palmitate uncouple AMPK
from autophagy in human aortic endothelial
cells. Am J Physiol Cell Physiol 2015; 308:
C249-263.
[26] Sun SY. Observation on the clinical effect of
Chaiqi decoction on 78 cases of metabolic
syndrome. Hebei Chinese Medicine 2014; 36:
1788-1789+1830.
[27] Wang Y, Liu J, Wang XX and Zhang LP. Effects
of Chaiqi decoction on glucose and lipid
metabolism and insulin resistance in rats
with metabolic syndrome. Journal of Beijing
University of Chinese Medicine 2015; 38: 597-
600.
[28] Liu J, Zhang LP, Wang Y, Wang XX, Zheng H
and Yan Y. Study on the mechanism of Chaiqi
decoction interfering metabolic syndrome.
Journal of Beijing University of Chinese
Medicine 2012; 35: 673-678+721.
[29] Chen LR, Liu Y, Zhang JY, Peng P and Zhang
LP. Study on the intervention mechanism
of Chaiqi decoction in the prevention and
treatment of vascular injury in rats with meta-
bolic syndrome. Journal of Chinese Medicine
Information 2017; 34: 39-44.
4920 Am J Transl Res 2020;12(9):4902-4922
[30] Dan HC and Baldwin AS. Differential involve-
ment of IkappaB kinases alpha and beta in
cytokine- and insulin-induced mammalian tar-
get of rapamycin activation determined by Akt.
J Immunol 2008; 180: 7582-7589.
[31] Zhou YD, Cao XQ, Liu ZH, Cao YJ, Liu CF, Zhang
YL and Xie Y. Rapamycin inhibits oxidized low
density lipoprotein uptake in human umbilical
vein endothelial cells via mTOR/NF-κB/LOX-1
pathway. PLoS One 2016; 11: e0146777.
[32] Zhou W and Ye S. Rapamycin improves insulin
resistance and hepatic steatosis in type 2 dia-
betes rats through activation of autophagy.
Cell Biol Int 2018; 42: 1282-1291.
[33] Bernardis LL and Patterson BD. Correlation
between ‘Lee index’ and carcass fat content
in weanling and adult female rats with hypo-
thalamic lesions. J Endocrinol 1968; 40: 527-
528.
[34] Kimura S, Noda T and Yoshimori T. Dissection
of the autophagosome maturation process by
a novel reporter protein, tandem fluorescent-
tagged LC3. Autophagy 2007; 3: 452-460.
[35] Iwashita H, Sakurai HT, Nagahora N, Ishiyama
M, Shioji K, Sasamoto K, Okuma K, Shimizu S
and Ueno Y. Small fluorescent molecules for
monitoring autophagic flux. FEBS Lett 2018;
592: 559-567.
[36] Kee HJ, Kim JR, Nam KI, Park HY, Shin S, Kim
JC, Shimono Y, Takahashi M, Jeong MH, Kim N,
Kim KK and Kook H. Enhancer of polycomb1,
a novel homeodomain only protein-binding
partner, induces skeletal muscle differentia-
tion. J Biol Chem 2007; 282: 7700-7709.
[37] Banerji V and Gibson SB. Targeting metabo-
lism and autophagy in the context of haemato-
logic malignancies. Int J Cell Biol 2012; 2012:
595976.
[38] Dow CA, Lincenberg GM, Greiner JJ, Stauffer
BL and DeSouza CA. Endothelial vasodilator
function in normal-weight adults with meta-
bolic syndrome. Appl Physiol Nutr Metab 2016;
41: 1013-1017.
[39] Xu Y. Transcriptional regulation of endothelial
dysfunction in atherosclerosis: an epigenetic
perspective. J Biomed Res 2014; 28: 47-52.
[40] Sena CM, Pereira AM and Seiça R. Endothelial
dysfunction - a major mediator of diabetic
vascular disease. Biochim Biophys Acta 2013;
1832: 2216-2231.
[41] Zhuhua Z, Zhiquan W, Zhen Y, Yixin N, Weiwei
Z, Xiaoyong L, Yueming L, Hongmei Z, Li Q and
Qing S. A novel mice model of metabolic syn-
drome: the high-fat-high-fructose diet-fed ICR
mice. Exp Anim 2015; 64: 435-442.
[42] Poudyal H, Panchal S and Brown L. Comparison
of purple carrot juice and β-carotene in a high-
carbohydrate, high-fat diet-fed rat model of the
metabolic syndrome. Br J Nutr 2010; 104:
1322-1332.
 Chaiqi decoction ameliorates vascular endothelial injury
[43] Wada T, Kenmochi H, Miyashita Y, Sasaki M,
Ojima M, Sasahara M, Koya D, Tsuneki H and
Sasaoka T. Spironolactone improves glucose
and lipid metabolism by ameliorating hepatic
steatosis and inflammation and suppressing
enhanced gluconeogenesis induced by high-
fat and high-fructose diet. Endocrinology 2010;
151: 2040-2049.
[44] Chidambaram J and Carani Venkatraman A.
Cissus quadrangularis stem alleviates insulin
resistance, oxidative injury and fatty liver dis-
ease in rats fed high fat plus fructose diet.
Food Chem Toxicol 2010; 48: 2021-2029.
[45] Lehnen AM, Rodrigues B, Irigoyen MC, De
Angelis K and Schaan BD. Cardiovascular
changes in animal models of metabolic syn-
drome. J Diabetes Res 2013; 2013: 761314.
[46] Li T, Lu X, Sun Y and Yang X. Effects of spinach
nitrate on insulin resistance, endothelial dys-
function markers and inflammation in mice
with high-fat and high-fructose consumption.
Food Nutr Res 2016; 60: 32010.
[47] Miles JM, Wooldridge D, Grellner WJ, Windsor
S, Isley WL, Klein S and Harris WS. Nocturnal
and postprandial free fatty acid kinetics in
normal and type 2 diabetic subjects: effects of
insulin sensitization therapy. Diabetes 2003;
52: 675-681.
[48] Lu Y, Qian L, Zhang Q, Chen B, Gui L, Huang D,
Chen G and Chen L. Palmitate induces apopto-
sis in mouse aortic endothelial cells and endo-
thelial dysfunction in mice fed high-calorie and
high-cholesterol diets. Life Sci 2013; 92: 1165-
1173.
[49] Titov VN. The excess of palmitic fatty acid in
food as main cause of lipoidosis of insulin-de-
pendent cells: Skeletal myocytes, cardio-myo-
cytes, periportal hepatocytes, kupffer macro-
phages and b-cells of pancreas. Klin Lab Diagn
2016; 61: 68-77.
[50] Aylett CH, Sauer E, Imseng S, Boehringer D,
Hall MN, Ban N and Maier T. Architecture of
human mTOR complex 1. Science 2016; 351:
48-52.
[51] Makki K, Taront S, Molendi-Coste O, Bouchaert
E, Neve B, Eury E, Lobbens S, Labalette M,
Duez H, Staels B, Dombrowicz D, Froguel P and
Wolowczuk I. Beneficial metabolic effects of
rapamycin are associated with enhanced regu-
latory cells in diet-induced obese mice. PLoS
One 2014; 9: e92684.
[52] Tianshu C, Congrong G, Zhiwei Z, Fei L, Ayu S,
Yuanbiao Z, Jing C and Ge J. Rapamycin com-
bined with α-cyanoacrylate contributes to in-
hibiting intimal hyperplasia in rat models. Arq
Bras Cardiol 2019; 112: 3-10.
[53] Ceylan-Isik AF, Dong M, Zhang Y, Dong F,
Turdi S, Nair S, Yanagisawa M and Ren J.
Cardiomyocyte-specific deletion of endothelin
4921 Am J Transl Res 2020;12(9):4902-4922
receptor A rescues aging-associated cardiac
hypertrophy and contractile dysfunction: role
of autophagy. Basic Res Cardiol 2013; 108:
335-63.
[54] Chambers metabolic syndrome, Rugo HS,
Litton JK and Meiller TF. Stomatitis associated
with mammalian target of rapamycin inhibi-
tion: a review of pathogenesis, prevention,
treatment, and clinical implications for oral
practice in metastatic breast cancer. J Am
Dent Assoc 2018; 149: 291-298.
[55] Hahn D, Hodson EM, Hamiwka LA, Lee VW,
Chapman JR, Craig JC and Webster AC. Target
of rapamycin inhibitors (TOR-I; sirolimus and
everolimus) for primary immunosuppression
in kidney transplant recipients. Cochrane
Database Syst Rev 2019; 12: Cd004290.
[56] Lloberas N, Cruzado JM, Franquesa M, Herrero-
Fresneda I, Torras J, Alperovich G, Rama I,
Vidal A and Grinyó JM. Mammalian target of
rapamycin pathway blockade slows progres-
sion of diabetic kidney disease in rats. J Am
Soc Nephrol 2006; 17: 1395-1404.
[57] Yang Y, Wang J, Qin L, Shou Z, Zhao J, Wang H,
Chen Y and Chen J. Rapamycin prevents early
steps of the development of diabetic nephrop-
athy in rats. Am J Nephrol 2007; 27: 495-502.
[58] Kolosova NG, Muraleva NA, Zhdankina AA,
Stefanova NA, Fursova AZ and Blagosklonny
MV. Prevention of age-related macular degen-
eration-like retinopathy by rapamycin in rats.
Am J Pathol 2012; 181: 472-477.
[59] Das A, Durrant D, Koka S, Salloum FN, Xi L and
Kukreja RC. Mammalian target of rapamycin
(mTOR) inhibition with rapamycin improves
cardiac function in type 2 diabetic mice: poten-
tial role of attenuated oxidative stress and
altered contractile protein expression. J Biol
Chem 2014; 289: 4145-4160.
[60] Reifsnyder PC, Doty R and Harrison DE.
rapamycin ameliorates nephropathy despite
elevating hyperglycemia in a polygenic mouse
model of type 2 diabetes, NONcNZO10/LtJ.
PLoS One 2014; 9: e114324.
[61] Wang S, Zhou SL, Min FY, Ma JJ, Shi XJ,
Bereczki E and Wu J. mTOR-mediated hyper-
phosphorylation of tau in the hippocampus is
involved in cognitive deficits in streptozotocin-
induced diabetic mice. Metab Brain Dis 2014;
29: 729-736.
[62] Gong FH, Ye YN, Li JM, Zhao HY and Li XK.
Rapamycin-ameliorated diabetic symptoms
involved in increasing adiponectin expression
in diabetic mice on a high-fat diet. Kaohsiung J
Med Sci 2017; 33: 321-326.
[63] Hartford CM and Ratain MJ. Rapamycin: some-
thing old, something new, sometimes bor-
rowed and now renewed. Clin Pharmacol Ther
2007; 82: 381-388.
 Chaiqi decoction ameliorates vascular endothelial injury
[64] Guo F, Li X, Peng J, Tang Y, Yang Q, Liu L, Wang
Z, Jiang Z, Xiao M, Ni C, Chen R, Wei D and
Wang GX. Autophagy regulates vascular endo-
thelial cell eNOS and ET-1 expression induced
by laminar shear stress in an ex vivo perfused
system. Ann Biomed Eng 2014; 42: 1978-
1988.
[65] DeMartino GN. Introduction to the thematic
minireview series: autophagy. J Biol Chem
2018; 293: 5384-5385.
[66] Xie K, Jin B, Li Y, Luo X, Zhu J, Ma D and Shi H.
Modulating autophagy improves cardiac func-
tion in a rat model of early-stage dilated car-
diomyopathy. Cardiology 2013; 125: 60-68.
[67] Maejima Y. The critical role of autophagy in
heart failure. Nihon Yakurigaku Zasshi 2018;
151: 100-105.
[68] Yan S and Xiu-ru G. Autophagy: a new target
for the treatment of atherosclerosis. Frontiers
in Laboratory Medicine 2018; S2542364-
918300487.
[69] Lin XL, Xiao WJ, Xiao LL and Liu MH. Molecular
mechanisms of autophagy in cardiac isch-
emia/reperfusion injury (Review). Mol Med
Rep 2018; 18: 675-683.
[70] Kuroki T, Isshiki K and King GL. Oxidative
stress: the lead or supporting actor in the
pathogenesis of diabetic complications. J Am
Soc Nephrol 2003; 14: S216-220.
[71] Zheng XT, Wu ZH, Wei Y, Dai JJ, Yu GF, Yuan F
and Ye LC. Induction of autophagy by salidro-
side through the AMPK-mTOR pathway pro-
tects vascular endothelial cells from oxidative
stress-induced apoptosis. Mol Cell Biochem
2017; 425: 125-138.
[72] Liu J, Wu J, Sun A, Sun Y, Yu X, Liu N, Dong S,
Yang F, Zhang L, Zhong X, Xu C, Lu F and Zhang
W. Hydrogen sulfide decreases high glucose/
palmitate-induced autophagy in endothelial
cells by the Nrf2-ROS-AMPK signaling path-
way. Cell Biosci 2016; 6: 33-50.
[73] Chen P, Liu H, Xiang H, Zhou J, Zeng Z, Chen R,
Zhao S, Xiao J, Shu Z, Chen S and Lu H. Palmitic
acid-induced autophagy increases reactive ox-
ygen species via the Ca(2+)/PKCα/NOX4 path-
way and impairs endothelial function in human
umbilical vein endothelial cells. Exp Ther Med
2019; 17: 2425-2432.
[74] Ye M, Qiu H, Cao Y, Zhang M, Mi Y, Yu J and
Wang C. Curcumin Improves palmitate-induced
insulin resistance in human umbilical vein
endothelial cells by maintaining proteostasis
in endoplasmic reticulum. Front Pharmacol
2017; 8: 148-60.
[75] Han J, Pan XY, Xu Y, Xiao Y, An Y, Tie L, Pan Y
and Li XJ. Curcumin induces autophagy to
protect vascular endothelial cell survival from
oxidative stress damage. Autophagy 2012; 8:
812-825.
4922 Am J Transl Res 2020;12(9):4902-4922
[76] Song J, Huang Y, Zheng W, Yan J, Cheng M,
Zhao R, Chen L, Hu C and Jia W. Resveratrol
reduces intracellular reactive oxygen species
levels by inducing autophagy through the
AMPK-mTOR pathway. Front Med 2018; 12:
697-706.
[77] Zhou X, Yang J, Zhou M, Zhang Y, Liu Y, Hou P,
Zeng X, Yi L and Mi M. Resveratrol attenuates
endothelial oxidative injury by inducing au-
tophagy via the activation of transcription fac-
tor EB. Nutr Metab (Lond) 2019; 16: 42-54.
[78] Geng FH, Li GH, Zhang X, Zhang P, Dong MQ,
Zhao ZJ, Zhang Y, Dong L and Gao F. Berberine
improves mesenteric artery insulin sensitivity
through up-regulating insulin receptor-mediat-
ed signalling in diabetic rats. Br J Pharmacol
2016; 173: 1569-1579.
[79] Cheraghi O, Abdollahpourasl M, Rezabakhsh A
and Rahbarghazi R. Distinct effects of royal
jelly on human endothelial cells under high
glucose condition. Iran J Pharm Res 2018; 17:
1361-1370.
[80] Zhao Q, Yang H, Liu F, Luo J, Zhao Q, Li X and
Yang Y. Naringenin exerts cardiovascular pro-
tective effect in a palmitate-induced human
umbilical vein endothelial cell injury model via
autophagy flux improvement. Mol Nutr Food
Res 2019; 63: e1900601.
[81] Wirth M, Joachim J and Tooze SA. Autopha-
gosome formation--the role of ULK1 and
Beclin1-PI3KC3 complexes in setting the
stage. Semin Cancer Biol 2013; 23: 301-309.
[82] Mizushima N, Yamamoto A, Hatano M, Koba-
yashi Y, Kabeya Y, Suzuki K, Tokuhisa T, Ohsumi
Y and Yoshimori T. Dissection of autophago-
some formation using Apg5-deficient mouse
embryonic stem cells. J Cell Biol 2001; 152:
657-668.
[83] Itakura E, Kishi-Itakura C and Mizushima N.
The hairpin-type tail-anchored SNARE syntaxin
17 targets to autophagosomes for fusion with
endosomes/lysosomes. Cell 2012; 151: 1256-
1269.
[84] Hebbachi A and Saggerson D. Acute regulation
of 5’-AMP-activated protein kinase by long-
chain fatty acid, glucose and insulin in rat pri-
mary adipocytes. Biosci Rep 2012; 33: 71-82.
[85] Ding WX. Uncoupling AMPK from autophagy: a
foe that hinders the beneficial effects of met-
formin treatment on metabolic syndrome-as-
sociated atherosclerosis? Focus on “glucose
and palmitate uncouple AMPK from autophagy
in human aortic endothelial cells”. Am J Physiol
Cell Physiol 2015; 308: C246-248.